Surviving winter on the Qinghai-Tibetan Plateau: Pikas suppress energy demands and exploit yak feces to survive winter.

The Qinghai-Tibetan Plateau, with low precipitation, low oxygen partial pressure, and temperatures routinely dropping below -30 °C in winter, presents several physiological challenges to its fauna. Yet it is home to many endemic mammalian species, including the plateau pika (Ochotona curzoniae). How these small animals that are incapable of hibernation survive the winter is an enigma. Measurements of daily energy expenditure (DEE) using the doubly labeled water method show that pikas suppress their DEE during winter. At the same body weight, pikas in winter expend 29.7% less than in summer, despite ambient temperatures being approximately 25 °C lower. Combined with resting metabolic rates (RMRs), this gives them an exceptionally low metabolic scope in winter (DEE/RMRt = 1.60 ± 0.30; RMRt is resting metabolic rate at thermoneutrality). Using implanted body temperature loggers and filming in the wild, we show that this is achieved by reducing body temperature and physical activity. Thyroid hormone (T3 and T4) measurements indicate this metabolic suppression is probably mediated via the thyroid axis. Winter activity was lower at sites where domestic yak (Bos grunniens) densities were higher. Pikas supplement their food intake at these sites by eating yak feces, demonstrated by direct observation, identification of yak DNA in pika stomach contents, and greater convergence in the yak/pika microbiotas in winter. This interspecific coprophagy allows pikas to thrive where yak are abundant and partially explains why pika densities are higher where domestic yak, their supposed direct competitors for food, are more abundant.

Large effective population size masks population genetic structure in Hirondellea amphipods within the deepest marine ecosystem, the Mariana Trench.

The examination of genetic structure in the deep-ocean hadal zone has focused on divergence between tectonic trenches to understand how environment and geography may drive species divergence and promote endemism. There has been little attempt to examine localized genetic structure within trenches, partly because of logistical challenges associated with sampling at an appropriate scale, and the large effective population sizes of species that can be sampled adequately may mask underlying genetic structure. Here we examine genetic structure in the superabundant amphipod Hirondellea gigas in the Mariana Trench at depths of 8126-10,545 m. RAD sequencing was used to identify 3182 loci containing 43,408 single nucleotide polymorphisms (SNPs) across individuals after stringent pruning of loci to prevent paralogous multicopy genomic regions being erroneously merged. Principal components analysis of SNP genotypes resolved no genetic structure between sampling locations, consistent with a signature of panmixia. However, discriminant analysis of principal components identified divergence between all sites driven by 301 outlier SNPs in 169 loci and significantly associated with latitude and depth. Functional annotation of loci identified differences between singleton loci used in analysis and paralogous loci pruned from the data set and also between outlier and nonoutlier loci, all consistent with hypotheses explaining the role of transposable elements driving genome dynamics. This study challenges the traditional perspective that highly abundant amphipods within a trench form a single panmictic population. We discuss the findings in relation to eco-evolutionary and ontogenetic processes operating in the deep sea, and highlight key challenges associated with population genetic analysis in nonmodel systems with inherent large effective population sizes and genomes.

Estimating demographic contributions to effective population size in an age-structured wild population experiencing environmental and demographic stochasticity.

A population's effective size (Ne ) is a key parameter that shapes rates of inbreeding and loss of genetic diversity, thereby influencing evolutionary processes and population viability. However, estimating Ne , and identifying key demographic mechanisms that underlie the Ne to census population size (N) ratio, remains challenging, especially for small populations with overlapping generations and substantial environmental and demographic stochasticity and hence dynamic age-structure. A sophisticated demographic method of estimating Ne /N, which uses Fisher's reproductive value to account for dynamic age-structure, has been formulated. However, this method requires detailed individual- and population-level data on sex- and age-specific reproduction and survival, and has rarely been implemented. Here, we use the reproductive value method and detailed demographic data to estimate Ne /N for a small and apparently isolated red-billed chough (Pyrrhocorax pyrrhocorax) population of high conservation concern. We additionally calculated two single-sample molecular genetic estimates of Ne to corroborate the demographic estimate and examine evidence for unobserved immigration and gene flow. The demographic estimate of Ne /N was 0.21, reflecting a high total demographic variance (σ2dg) of 0.71. Females and males made similar overall contributions to σ2dg. However, contributions varied among sex-age classes, with greater contributions from 3 year-old females than males, but greater contributions from ≥5 year-old males than females. The demographic estimate of Ne was ~30, suggesting that rates of increase of inbreeding and loss of genetic variation per generation will be relatively high. Molecular genetic estimates of Ne computed from linkage disequilibrium and approximate Bayesian computation were approximately 50 and 30, respectively, providing no evidence of substantial unobserved immigration which could bias demographic estimates of Ne . Our analyses identify key sex-age classes contributing to demographic variance and thus decreasing Ne /N in a small age-structured population inhabiting a variable environment. They thereby demonstrate how assessments of Ne can incorporate stochastic sex- and age-specific demography and elucidate key demographic processes affecting a population's evolutionary trajectory and viability. Furthermore, our analyses show that Ne for the focal chough population is critically small, implying that management to re-establish genetic connectivity may be required to ensure population viability.

The role of parasite-driven selection in shaping landscape genomic structure in red grouse (Lagopus lagopus scotica).

Landscape genomics promises to provide novel insights into how neutral and adaptive processes shape genome-wide variation within and among populations. However, there has been little emphasis on examining whether individual-based phenotype-genotype relationships derived from approaches such as genome-wide association (GWAS) manifest themselves as a population-level signature of selection in a landscape context. The two may prove irreconcilable as individual-level patterns become diluted by high levels of gene flow and complex phenotypic or environmental heterogeneity. We illustrate this issue with a case study that examines the role of the highly prevalent gastrointestinal nematode Trichostrongylus tenuis in shaping genomic signatures of selection in red grouse (Lagopus lagopus scotica). Individual-level GWAS involving 384 SNPs has previously identified five SNPs that explain variation in T. tenuis burden. Here, we examine whether these same SNPs display population-level relationships between T. tenuis burden and genetic structure across a small-scale landscape of 21 sites with heterogeneous parasite pressure. Moreover, we identify adaptive SNPs showing signatures of directional selection using F(ST) outlier analysis and relate population- and individual-level patterns of multilocus neutral and adaptive genetic structure to T. tenuis burden. The five candidate SNPs for parasite-driven selection were neither associated with T. tenuis burden on a population level, nor under directional selection. Similarly, there was no evidence of parasite-driven selection in SNPs identified as candidates for directional selection. We discuss these results in the context of red grouse ecology and highlight the broader consequences for the utility of landscape genomics approaches for identifying signatures of selection.

Evidence of the phenotypic expression of a lethal recessive allele under inbreeding in a wild population of conservation concern.

Deleterious recessive alleles that are masked in outbred populations are predicted to be expressed in small, inbred populations, reducing both individual fitness and population viability. However, there are few definitive examples of phenotypic expression of lethal recessive alleles under inbreeding conditions in wild populations. Studies that demonstrate the action of such alleles, and infer their distribution and dynamics, are required to understand their potential impact on population viability and inform management responses. The Scottish population of red-billed choughs (Pyrrhocorax pyrrhocorax), which currently totals <60 breeding pairs and is of major conservation concern, has recently been affected by lethal blindness in nestlings. We used family data to show that the pattern of occurrence of blindness within and across affected families that produced blind nestlings was exactly 0·25, matching that expected given a single-locus autosomal lethal recessive allele. Furthermore, the observed distribution of blind nestlings within affected families did not differ from that expected given Mendelian inheritance of such an allele. Relatedness estimates showed that individuals from affected families were not more closely related to each other than they were to individuals from unaffected families that did not produce blind nestlings. Blind individuals tended to be less heterozygous than non-blind individuals, as expected if blindness was caused by the expression of a recessive allele under inbreeding. However, there was no difference in the variance in heterozygosity estimates, suggesting that some blind individuals were relatively outbred. These results suggest carriers of the blindness allele may be widely distributed across contemporary families rather than restricted to a single family lineage, implying that the allele has persisted across multiple generations. Blindness occurred at low frequency (affecting 1·6% of observed nestlings since 1981). However, affected families had larger initial brood sizes than unaffected families. Such high fecundity of carriers of a lethal recessive allele might reflect overdominance, potentially reducing purging and increasing allele persistence probability. We thereby demonstrate the phenotypic expression of a lethal recessive allele in a wild population of conservation concern, and provide a general framework for inferring allele distribution and persistence and informing management responses.

Isolation and Characterization of Microsatellite DNA Markers in the Deep-Sea Amphipod Paralicella tenuipes by Illumina MiSeq Sequencing.

Here, we describe the development of 16 polymorphic microsatellite markers using an Illumina MiSeq sequencing approach in the deep-sea amphipod Paralicella tenuipes A total of 25 577 844 DNA sequences were filtered for microsatellite motifs of which 197 873 sequences were identified. From these sequences, 64 had sufficient flanking regions for primer design and 16 of these loci were polymorphic. Between 5 and 30 alleles were detected per locus, with an average of 13.63 alleles per locus, across a total of 120 individuals from 5 separate deep sea trenches from the Pacific Ocean. For the 16 loci, observed and expected heterozygosity values ranged from 0.116 to 0.414 and 0.422 to 0.820, respectively, with one locus displaying significant deviation from Hardy-Weinberg equilibrium. The microsatellite loci that have been isolated and described here are the first molecular markers developed for deep sea amphipods and will be invaluable for elucidating the genetic population structure and the extent of connectivity between deep ocean trenches.

Genome-wide association and genome partitioning reveal novel genomic regions underlying variation in gastrointestinal nematode burden in a wild bird.

Identifying the genetic architecture underlying complex phenotypes is a notoriously difficult problem that often impedes progress in understanding adaptive eco-evolutionary processes in natural populations. Host-parasite interactions are fundamentally important drivers of evolutionary processes, but a lack of understanding of the genes involved in the host's response to chronic parasite insult makes it particularly difficult to understand the mechanisms of host life history trade-offs and the adaptive dynamics involved. Here, we examine the genetic basis of gastrointestinal nematode (Trichostrongylus tenuis) burden in 695 red grouse (Lagopus lagopus scotica) individuals genotyped at 384 genome-wide SNPs. We first use genome-wide association to identify individual SNPs associated with nematode burden. We then partition genome-wide heritability to identify chromosomes with greater heritability than expected from gene content, due to harbouring a multitude of additive SNPs with individually undetectable effects. We identified five SNPs on five chromosomes that accounted for differences of up to 556 worms per bird, but together explained at best 4.9% of the phenotypic variance. These SNPs were closely linked to genes representing a range of physiological processes including the immune system, protein degradation and energy metabolism. Genome partitioning indicated genome-wide heritability of up to 29% and three chromosomes with excess heritability of up to 4.3% (total 8.9%). These results implicate SNPs and novel genomic regions underlying nematode burden in this system and suggest that this phenotype is somewhere between being based on few large-effect genes (oligogenic) and based on a large number of genes with small individual but large combined effects (polygenic).

Fine-scale population epigenetic structure in relation to gastrointestinal parasite load in red grouse (Lagopus lagopus scotica).

Epigenetic modification of cytosine methylation states can be elicited by environmental stresses and may be a key process affecting phenotypic plasticity and adaptation. Parasites are potent stressors with profound physiological and ecological effects on their host, but there is little understanding in how parasites may influence host methylation states. Here, we estimate epigenetic diversity and differentiation among 21 populations of red grouse (Lagopus lagopus scotica) in north-east Scotland and test for association of gastrointestinal parasite load (caecal nematode Trichostrongylus tenuis) with hepatic genome-wide and locus-specific methylation states. Following methylation-sensitive AFLP (MSAP), 129 bands, representing 73 methylation-susceptible and 56 nonmethylated epiloci, were scored across 234 individuals. The populations differed significantly in genome-wide methylation levels and were also significantly epigenetically (F(SC) = 0.0227; P < 0.001) and genetically (F(SC) = 0.0058; P < 0.001) differentiated. Parasite load was not associated with either genome-wide methylation levels or epigenetic differentiation. Instead, we found eight disproportionately differentiated epilocus-specific methylation states (F(ST) outliers) using bayescan software and significant positive and negative association of 35 methylation states with parasite load from bespoke generalized estimating equations (GEE), simple logistic regression (sam) and Bayesian environmental analysis (bayenv2). Following Sanger sequencing, genome mapping and geneontology (go) annotation, some of these epiloci were linked to genes involved in regulation of cell cycle, signalling, metabolism, immune system and notably rRNA methylation, histone acetylation and small RNAs. These findings demonstrate an epigenetic signature of parasite load in populations of a wild bird and suggest intriguing physiological effects of parasite-associated cytosine methylation.

Eco-evolutionary dynamics in response to selection on life-history.

Understanding the consequences of environmental change on ecological and evolutionary dynamics is inherently problematic because of the complex interplay between them. Using invertebrates in microcosms, we characterise phenotypic, population and evolutionary dynamics before, during and after exposure to a novel environment and harvesting over 20 generations. We demonstrate an evolved change in life-history traits (the age- and size-at-maturity, and survival to maturity) in response to selection caused by environmental change (wild to laboratory) and to harvesting (juvenile or adult). Life-history evolution, which drives changes in population growth rate and thus population dynamics, includes an increase in age-to-maturity of 76% (from 12.5 to 22 days) in the unharvested populations as they adapt to the new environment. Evolutionary responses to harvesting are outweighed by the response to environmental change (~ 1.4 vs. 4% change in age-at-maturity per generation). The adaptive response to environmental change converts a negative population growth trajectory into a positive one: an example of evolutionary rescue.

Selection maintains MHC diversity through a natural population bottleneck.

A perceived consequence of a population bottleneck is the erosion of genetic diversity and concomitant reduction in individual fitness and evolutionary potential. Although reduced genetic variation associated with demographic perturbation has been amply demonstrated for neutral molecular markers, the effective management of genetic resources in natural populations is hindered by a lack of understanding of how adaptive genetic variation will respond to population fluctuations, given these are affected by selection as well as drift. Here, we demonstrate that selection counters drift to maintain polymorphism at a major histocompatibility complex (MHC) locus through a population bottleneck in an inbred island population of water voles. Before and after the bottleneck, MHC allele frequencies were close to balancing selection equilibrium but became skewed by drift when the population size was critically low. MHC heterozygosity generally conformed to Hardy-Weinberg expectations except in one generation during the population recovery where there was a significant excess of heterozygous genotypes, which simulations ascribed to strong differential MHC-dependent survival. Low allelic diversity and highly skewed frequency distributions at microsatellite loci indicated potent genetic drift due to a strong founder affect and/or previous population bottlenecks. This study is a real-time examination of the predictions of fundamental evolutionary theory in low genetic diversity situations. The findings highlight that conservation efforts to maintain the genetic health and evolutionary potential of natural populations should consider the genetic basis for fitness-related traits, and how such adaptive genetic diversity will vary in response to both the demographic fluctuations and the effects of selection.

Genetic differentiation among North Atlantic killer whale populations.

Population genetic structure of North Atlantic killer whale samples was resolved from differences in allele frequencies of 17 microsatellite loci, mtDNA control region haplotype frequencies and for a subset of samples, using complete mitogenome sequences. Three significantly differentiated populations were identified. Differentiation based on microsatellite allele frequencies was greater between the two allopatric populations than between the two pairs of partially sympatric populations. Spatial clustering of individuals within each of these populations overlaps with the distribution of particular prey resources: herring, mackerel and tuna, which each population has been seen predating. Phylogenetic analyses using complete mitogenomes suggested two populations could have resulted from single founding events and subsequent matrilineal expansion. The third population, which was sampled at lower latitudes and lower density, consisted of maternal lineages from three highly divergent clades. Pairwise population differentiation was greater for estimates based on mtDNA control region haplotype frequencies than for estimates based on microsatellite allele frequencies, and there were no mitogenome haplotypes shared among populations. This suggests low or no female migration and that gene flow was primarily male mediated when populations spatially and temporally overlap. These results demonstrate that genetic differentiation can arise through resource specialization in the absence of physical barriers to gene flow.

Plasticity is a locally adapted trait with consequences for ecological dynamics in novel environments.

Phenotypic plasticity is predicted to evolve in more variable environments, conferring an advantage on individual lifetime fitness. It is less clear what the potential consequences of that plasticity will have on ecological population dynamics. Here, we use an invertebrate model system to examine the effects of environmental variation (resource availability) on the evolution of phenotypic plasticity in two life history traits-age and size at maturation-in long-running, experimental density-dependent environments. Specifically, we then explore the feedback from evolution of life history plasticity to subsequent ecological dynamics in novel conditions. Plasticity in both traits initially declined in all microcosm environments, but then evolved increased plasticity for age-at-maturation, significantly so in more environmentally variable environments. We also demonstrate how plasticity affects ecological dynamics by creating founder populations of different plastic phenotypes into new microcosms that had either familiar or novel environments. Populations originating from periodically variable environments that had evolved greatest plasticity had lowest variability in population size when introduced to novel environments than those from constant or random environments. This suggests that while plasticity may be costly it can confer benefits by reducing the likelihood that offspring will experience low survival through competitive bottlenecks in variable environments. In this study, we demonstrate how plasticity evolves in response to environmental variation and can alter population dynamics-demonstrating an eco-evolutionary feedback loop in a complex animal moderated by plasticity in growth.

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach.

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance.

Extravagant ornaments evolved to advertise their bearers' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties.

Spatio-temporal variation in the strength and mode of selection acting on major histocompatibility complex diversity in water vole (Arvicola terrestris) metapopulations.

Patterns of spatio-temporal genetic variation at a class II major histocompatibility complex (MHC) locus and multiple microsatellite loci were analysed within and between three water vole metapopulations in Scotland, UK. Comparisons of MHC and microsatellite spatial genetic differentiation, based on standardised tests between two demographically asynchronous zones within a metapopulation, suggested that spatial MHC variation was affected by balancing selection, directional selection and random genetic drift, but that the relative effects of these microevolutionary forces vary temporally. At the metapopulation level, between-year differentiation for MHC loci was significantly correlated with that of microsatellites, signifying that neutral factors such as migration and drift were primarily responsible for overall temporal genetic change at the metapopulation scale. Between metapopulations, patterns of genetic differentiation implied that, at large spatial scales, MHC variation was primarily affected by directional selection and drift. Levels of MHC heterozygosity in excess of Hardy-Weinberg expectations were consistent with overdominant balancing selection operating on MHC variation within metapopulations. However, this effect was not constant among all samples, indicating temporal variation in the strength of selection relative to other factors. The results highlight the benefit of contrasting variation at MHC with neutral markers to separate the effects of stochastic and deterministic microevolutionary forces, and add to a growing body of evidence showing that the mode and relative strength of selection acting on MHC diversity varies both spatially and temporally.

Landscape barriers reduce gene flow in an invasive carnivore: geographical and local genetic structure of American mink in Scotland.

To be effective, management programmes geared towards halting or reversing the spread of invasive species must focus on defined and defensible areas. This requires knowledge of the dispersal of non-native species targeted for control to better understand invasion and recolonisation scenarios. We investigated the genetic structure of invasive American mink (Neovison vison) in Scotland, and incorporated landscape genetic approaches to examine resultant patterns in relation to geographical features that may influence dispersal. Populations of mink sampled from 10 sites in two regions (Argyll and Northeast Scotland) show a distinct genetic structure. First, the majority of pairwise population comparisons yielded F(ST) values that were significantly greater than zero. Second, AMOVA revealed that most of the genetic variance was attributable to differences among regions. Assignment tests placed 89 or more of individuals into their sampled region. Bayesian clustering methods grouped samples into two clusters according to their region of origin. Wombling approach identified the Cairngorms Mountains as a major impediment to gene flow between the regions. Mantel pairwise correlations between genetic and geographical distances estimated as least-cost distance assuming a linear increase in the cost of movement with increasing elevation were higher than Euclidean distances or distance along waterways. Spatial autocorrelation analyses revealed stronger spatial structuring for females than for males. These results suggest that gene flow by American mink is restricted by landscape features (mountain ranges) and that eradication attempt should in the first instance break down the connectivity between management units separated by mountains.

Ecological, morphological and genetic divergence of sympatric North Atlantic killer whale populations.

Ecological divergence has a central role in speciation and is therefore an important source of biodiversity. Studying the micro-evolutionary processes of ecological diversification at its early stages provides an opportunity for investigating the causative mechanisms and ecological conditions promoting divergence. Here we use morphological traits, nitrogen stable isotope ratios and tooth wear to characterize two disparate types of North Atlantic killer whale. We find a highly specialist type, which reaches up to 8.5 m in length and a generalist type which reaches up to 6.6 m in length. There is a single fixed genetic difference in the mtDNA control region between these types, indicating integrity of groupings and a shallow divergence. Phylogenetic analysis indicates this divergence is independent of similar ecological divergences in the Pacific and Antarctic. Niche-width in the generalist type is more strongly influenced by between-individual variation rather than within-individual variation in the composition of the diet. This first step to divergent specialization on different ecological resources provides a rare example of the ecological conditions at the early stages of adaptive radiation.

Discrimination of Culicoides midge larvae using multiplex polymerase chain reaction assays based on DNA sequence variation at the mitochondrial cytochrome C oxidase I gene.

The recent spread of Bluetongue disease in northwestern Europe has indicated the ability of Palaearctic Culicoides species to vector the disease. Because the different midge species vary in their ability to harbor and transmit the Bluetongue virus, quick and reliable identification is necessary to resolve the species composition of midge communities, both adult and larval, at any place at any given time point. Given that morphological identification of Culicoides species is problematic, we developed three multiplex polymerase chain reaction (PCR) assays that facilitate high-throughput analysis of midge specimens. One assay distinguishes between species of the so-called Culicoides obsoletus s.l. complex (including C. dewulfi), whereas two assays facilitate differentiation of species of the Culicoides pulicaris s.l. complex. These assays yield two PCR products: one species-specific and one generic band. We show the application of the assays in the analysis of Culicoides larvae from three different farms in northeast Scotland.

Author Correction: Signatures of balancing selection in toll-like receptor (TLRs) genes - novel insights from a free-living rodent.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Spatial distribution of mitochondrial and microsatellite DNA variation in Daubenton's bat within Scotland.

Daubenton's bat (Myotis daubentonii) is a known reservoir for European bat lyssavirus type 2 (EBLV-2). An appreciation of the potential for epidemiological spread and disease risk requires an understanding of the dispersal of the primary host, and any large-scale geographical barriers that may impede gene flow. The spatial pattern of microsatellite and mitochondrial DNA variation was examined to infer patterns of dispersal of bats among 35 populations across Scotland. DNA sequence variation at the mitochondrial control region and ND1 genes revealed two distinct phylogeographical clades, with generally nonoverlapping geographical distributions except for a small number of populations where both matrilines were found in sympatry. Such discontinuity suggests that Scotland was recolonized twice following the retreat of the Pleistocene ice sheet with little subsequent matrilineal introgression. However, eight microsatellite loci showed low levels of genetic divergence among populations, even between populations from the two distinct mitochondrial DNA clades. An overall, macrogeographical genetic isolation-by-distance pattern was observed, with high levels of gene flow among local populations. Apparently contrasting patterns of mitochondrial and microsatellite divergence at different scales could be explained by sex-specific differences in gene flow at large scales.